Cloning and expression of human islet glutamic acid decarboxylase autoantigen

ABSTRACT

Human pancreatic islet cell glutamic acid decarboxylase (GAD), an autoantigen involved in the development of insulin-dependent diabetes mellitus (IDDM), has been cloned, sequenced and expressed by recombinant means. Recombinant human islet cell GAD polypeptides and antibodies specific to the GAD polypeptides can be used in methods of diagnosis and treatment, including use in immunoadsorptive therapy and the induction of immune tolerance.

GOVERNMENT SUPPORT

This invention was made with government support under grant numbersDK26190, DK33873, and DK41801 awarded by the National Institutes ofHealth/Juvenile Diabetes Foundation. The government has certain rightsin the invention.

RELATED CASES

This application is a continuation of U.S. application Ser. No.07/883,492, filed May 15, 1992, now abandoned, which is a continuationof U.S. application Ser. No. 07/702,162, filed May 15, 1991, which isincorporated herein by reference.

BACKGROUND OF THE INVENTION

γ-Aminobutyric acid (GABA) is a major inhibitory neurotransmitter of themammalian central nervous system. The rate-limiting step in GABAbiosynthesis is the decarboxylation of L-glutamic acid by glutamic aciddecarboxylase (GAD). Little is known with certainty regarding theregulation of GAD activity or the expression of GAD genes. Despite itswide distribution in the brain, GAD protein is present in very smallquantities and is very difficult to purify to homogeneity.

Studies of GAD have been hindered by the existence of multiple forms ofthe enzyme, which differ in molecular weight, kinetic properties,sequence (when known), and hydrophobic properties. For example, thepresence of three different forms of GAD in porcine brain has beenreported (Spink et al., J. Neurochem. 40:1113-1119 (1983)), as well asfour forms in rat brain (Spink et al., Brain Res. 421:235-244 (1987)). Amouse brain GAD (Huang et al., Proc. Natl. Acad. Sci. USA 87:8491-8495(1990)) and a GAD clone isolated from feline brain Kobayashi et al., J.Neurosci. 7:2768-2772 (1987)) have also been reported. At least twoisomers of GAD have been reported in human brain. Chang and Gottlieb, J.Neurosci. 8:2123-2130 (1988).

Further complicating the characterization of distinct GAD isozymes isthe fact that GADs are also found in tissues outside of the brain, whichGADs have varying degrees of homology with brain GADs. For example, GADsare also expressed in germ cells of the testis, as has been reported forseveral different species, e.g., rat (Persson et al., in Perspectives ofAndrology, M. Serio, (ed.), p. 129-138, Raven Press, New York (1989)),and in human, guinea pig, monkey, and mouse testis (Perrson et al., Mol.Cell. Biol. 10:4701-4711 (1990). The human testis GAD was shown to havea relatively high degree of overall nucleotide sequence homology to thefeline brain GAD. The presence of GAD in the pancreas has also beendescribed. Okada et al., Science 194:620-622 (1976), Garry et al, J.Histochem. Cytochem. 36:573-580 (1988), and Vincent et al.,Neuroendocrin. 36:197-204 (1983).

A rare neurological disorder, termed Stiff-Man Syndrome (SMS), isassociated with the presence of autoantibodies to GABA-secretingneurons. The predominant autoantigen for the autoantibodies in SMS hasrecently been shown to be a brain GAD. Solimena et al., N. Engl. J. Med.318:1012-1020 (1988) and Solimena et al., N. Engl. J. Med. 322:1555-1560(1990).

A small proportion of patients with insulin-dependent diabetes mellitus(IDDM) also develop SMS. It has been speculated that autoimmunemechanisms may precipitate the clinical onset of IDDM. The destructionof pancreatic β(beta)-cells in the islets of Langerhans typicallyprecedes IDDM. This destruction is believed to be mediated by a massiveinfiltration by lymphocytes into the islets, and by the presence ofcirculating autoantibodies to the β-cells.

A major target of autoantibodies associated with the development of IDDMis a pancreatic β-cell antigen of relative molecular mass 64,000 (64K).Baekkeskov et al., J. Clin. Invest. 79:926-934 (1987). Antibodies to the64K antigen are present in greater than about 80% of newly diagnosedpatients and have been detected up to several years before clinicalonset of IDDM, concomitant with a gradual loss of β-cells. The 64Kantigen has recently been identified as GAD. Baekkeskov et al., Nature347:151-156 (1990).

As with brain GADs, the purification of native human islet cell GAD hasa number of disadvantages. Human islet GAD is only a trace protein andit would be impractical to isolate useful quantities from naturalsources. For example, purification of native human islet cell GAD wouldrequire a large number of human pancreata, which itself poses asubstantial obstacle. Further, purification from human tissues carriesthe risk of co-purifying infective agents such as the hepatitis viruses,retroviruses such as HIV-1 and HIV-2, and other viral agents. Not onlydoes this present the possibility of infecting recipients of therapeuticproducts with such agents, but raises significant concerns among workerswho manufacture and test these products as well as those who will usethe products in diagnostic laboratories.

There is a need in the art, therefore, for safe methods of producingrelatively large amounts of pure preparations of human islet cell GADpolypeptides. These polypeptides would be useful as, inter alia,therapeutic agents in the treatment of GAD-related diseases, such asIDDM, and in the diagnosis and monitoring of these diseases. Quiteremarkably, the present invention fulfills these and other related needsthrough the use of recombinant DNA technology, thus eliminating theproblem of viral contamination and providing commercially feasiblequantities of human islet cell GAD polypeptides.

SUMMARY OF THE INVENTION

The present invention provides the ability to produce human islet cellGAD polypeptides by recombinant or synthetic means. The human islet GADpolypeptides so produced may or may not have the biological activity ofthe native enzyme, depending on the intended use. Accordingly, isolatedand purified polynucleotides are described which code for the humanislet GAD polypeptides, where the polynucleotides may be in the form ofDNA, such as cDNA or genomic DNA, or RNA. Based on these sequencesprobes can be designed for hybridization to identify these and relatedgenes or transcription products thereof which encode human islet cellGAD.

In related embodiments the invention concerns DNA constructs whichcomprise a transcriptional promoter, a DNA sequence which encodes thehuman islet cell GAD polypeptide, and optionally a transcriptionalterminator, each operably linked for expression of the GAD polypeptide.The constructs are preferably used to transfect or transform host cells,preferably eukaryotic cells, more preferably mammalian or yeast cells.For large scale production the expressed human islet cell GADpolypeptides can be isolated from the cells by, for example,immunoaffinity purification. Small synthetic peptides can also beprepared which immunologically mimic GAD antigenic determinants or otherregions of the GAD molecule.

The GAD polypeptides of the invention can also be used therapeutically,for example, to remove autoantibodies to the GAD islet cellautoantigens, conveniently by extracorporeal immunoadsorptive means. TheGAD polypeptides described herein also can be formulated andadministered as pharmaceutical compositions, especially when used toinduce immunological tolerance in individuals predisposed to developingor already afflicted by disease caused by or related to autoantibodiesto GAD autoantigens, such as in IDDM.

In other embodiments the GAD polypeptides find use as diagnosticreagents, detecting and/or quantitating the level of GAD autoantibodiesin an individual of interest. These diagnostic methods and compositionscan be used in conjunction with therapeutic approaches to GAD-relateddiseases, particularly the treatment of IDDM.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram of partial human islet GAD clones;

FIG. 2 shows the nucleotide sequence and predicted primary structure ofhuman islet GAD, where nucleotides and amino acids are numbered at theleft ends of the lines; and

FIG. 3 is a hydropathy plot of human islet GAD and rat and cat brainGAD.

DESCRIPTION OF THE SELECTED EMBODIMENTS

Human pancreatic islet cell GAD is an enzyme that catalyzes thesynthesis of GABA. The present invention provides isolated nucleotidesequences of human islet cell GAD, thereby providing for the ultimateexpression of human islet cell GAD polypeptides. Recombinant DNAexpression systems provide convenient means for obtaining largequantities of recombinant human islet cell GAD and fragments thereof inrelatively pure form.

The invention also provides recombinant human islet cell GADpolypeptides. By "recombinant" is meant a polypeptide produced by arecombinant expression system and typically free of native endogenoussubstances. By "polypeptide" is meant to include sequences of at leastabout six amino acids, typically 10 to 25, and up to 100-200 amino acidsor more, including up to the entire human islet GAD protein. When thepolypeptide comprises the entire GAD protein, the polypeptides will besubstantially homologous to the entire human islet cell GAD sequence asdisclosed in SEQ. ID. NO. 1 and FIG. 2. By "substantially homologous" ismeant to include sequences which have at least about 85% homology,preferably at least 90%, and more preferably at least about 95% or morehomology to the amino acid sequence of the human islet cell GADsequence(s) of the invention and still retain at least some biologicalactivity of the native GAD. By "biological activity" is meant theability to catalyze the decarboxylation of L-glutamic acid, tospecifically bind antibodies which bind to the native human islet cellGAD protein (i.e., autoantibodies to human islet cell GAD), and/or toelicit antibodies which also bind to the native protein.

When the polypeptide of the invention comprises less than the entire GADprotein, the polypeptide will preferably be substantially homologous toa portion of at least about 6, sometimes 10, more usually at least about15 amino acids of a variable region of the GAD protein. That is, certainsequence domains are variable, differing at least about 15%, moretypically at least about 20%, from analogous regions of GADs of othertissues and/or species, while other regions of the human islet cell GADare identical or nearly identical to other GADS, and thus representconserved regions. The conserved and variable sequence regions of thehuman islet cell GAD of the present invention can be determined bytechniques known to the skilled artisan, such as sequence alignmenttechniques, e.g., using the Sequence Analysis Software Package of theGenetics Computer Group, University of Wisconsin Biotechnology Center,1710 University Avenue, Madison, Wis. 53705 (Devereux, Nuc. Acids. Res.12:387-396 (1984). Homology is determined by attaining optimal alignmentof the present GAD sequence with, e.g., brain GAD sequences of rat andcat, as disclosed in Julien et al., J. Neurochem. 54:703-705 (1990),incorporated herein by reference.

For example, in reference to FIG. 2, human islet GAD variable regiondomains, when compared to the amino acid sequence of cat and rat, areidentified at the N-terminal residues 1-91, and at positions 137-171,405-431, and 511-540. Epitopes which comprise at least a portion ofvariable region domain, typically at least about six contiguous aminoacids from the variable region and often ten or more residues, may serveas human islet cell GAD-specific markers.

As will be appreciated by those skilled in the art, the invention alsoincludes those polypeptides having slight variations in amino acidsequences or other properties. Such variations may arise naturally asallelic variations (e.g., due to genetic polymorphism) or may beproduced by human intervention (e.g., by mutagenesis of cloned DNAsequences), such as induced point, deletion, insertion and substitutionmutants. Minor changes in amino acid sequence are generally preferred,such as conservative amino acid replacements, small internal deletionsor insertions, and additions or deletions at the ends of the molecules.Substitutions may be designed based on, for example, the model ofDayhoff, et. al. (in Atlas of Protein Sequence and Structure 1978, Nat'lBiomed. Res. Found., Washington, D.C.). These modifications can resultin changes in the amino acid sequence, provide silent mutations, modifya restriction site, or provide other specific mutations. Thepolypeptides may comprise one or more selected antigenic determinants ofhuman islet GAD, possess catalytic activity exhibited by native GADprotein or alternatively lack such activity, mimic GAD binding regions,or the like.

Nucleic acid sequences encoding human islet cell GAD as described hereincan be cloned directly from human cell sources that express the enzyme.Preferred sources include human pancreatic islet cells, but neuronswhich secrete GABA may also serve as a source for the homologous humanbrain GAD. The nucleotide and amino acid sequences provided by thepresent invention can also be used to identify and clone other tissuespecific human GADs, as described further below. Useful nucleic acidsequences for cloning and expressing GAD sequences include mRNA, genomicDNA and cDNA, although for expression cDNAs are generally preferredbecause they lack introns that may interfere with expression.

To obtain a human islet GAD clone or other human GAD clone which issubstantially homologous to the human islet GAD, a cDNA library preparedfrom, e.g., human pancreatic islet cells, is screened with labeledprobes from the human islet GAD sequences provided herein, or fromhomologous sequence regions of, e.g., mouse brain GAD (Katarova et al.,Eur. J. Neurosci. 2:190-202 (1990)), cat brain GAD (Kobayashi et al. J.Neurosci. 7:2768-2772 (1987)), or rat brain GAD (Julien et al., J.Neurochem. 54:703-705 (1990)), each of which is incorporated herein byreference. An oligo-dT primed cDNA library can be constructed withpolyA⁺ RNA purified from human pancreatic islet cells or from othertissues/cells as desired. The library is screened with, e.g., antibodiesto homologous GAD and/or labeled probes. Partial clones may be used asprobes in additional screening until the complete coding sequence isobtained. If necessary, partial clones are joined in the correct readingframe to construct the complete coding sequence. Joining is achieved bydigesting clones with appropriate restriction endonucleases and joiningthe fragments enzymatically in the proper orientation. Depending on thefragments and the particular restriction endonucleases chosen, it may benecessary to remove unwanted DNA sequences through a "loop out" processof deletion mutagenesis or through a combination of restrictionendonuclease cleavage and mutagenesis. It is preferred that theresultant sequence be in the form of a continuous open reading frame,that is, that it lack intervening sequences (introns).

Representative human cDNA islet GAD clones isolated as described hereincan be combined to give the full coding sequence for the human isletcell GAD. For example, two clones, designated pHIG1.9 and pHIG11, arecombined to give the full coding sequence and 3'-untranslated sequences,as shown in FIG. 2, SEQ. ID. NO. 1. This clone has a polyadenylationsequence upstream of a poly A sequence at the 3' end. It will be readilyunderstood by those skilled in the art that due to the degeneracy of thecode, there can be considerable variation in a nucleotide sequence whichencodes the same amino acid sequence, or which sequence encodes a mutantsequence which varies from a reference sequence, such as that of SEQ.ID. NO 1 and FIG. 2. In the case of a mutant sequence, which mutantshould be substantially homologous to at least a portion of thereference sequence, the net effect of the mutation(s) should not produceadverse functional differences between the native and the mutantsequences. The identity of a human islet GAD clone can be confirmed by,for example, in vitro translation and subsequent immunoreactivity andmigration on an SDS-polyacrylamide gel, sequencing, or by appropriateenzymatic activity, e.g., catalyzing the synthesis of γ-aminobutyricacid. GAD catalytic activity can be assayed by CO₂ and/or GABA ethods asdescribed in, e.g., Chude and Wu, J. Neurochem. 27:83-86 (1976).Briefly, the assays can employ, e.g., L- U-¹⁴ C!glutamate as a substrateand the amounts of ¹⁴ CO₂ and ¹⁴ C!GABA formed are determined.

With the nucleotide and deduced amino acid sequences of human islet GADprovided herein, genomic or cDNA sequences encoding other human GADs maybe obtained from libraries prepared from other cells and tissuesaccording to known procedures. For instance, using oligonucleotideprobes derived from human islet cell GAD sequences, generally of atleast about fourteen nucleotides and up to twenty-five or morenucleotides in length, DNA sequences encoding GADs of other cells, suchas isozymes from beta cells, testis, or neurons, may be obtained. Again,if partial clones are obtained, it is necessary to join them in properreading frame to produce a full length clone, using such techniques asendonuclease cleavage, ligation and loopout mutagenesis.

For expression, a DNA sequence encoding human islet GAD is inserted intoa suitable expression vector, which in turn is used to transform ortransfect appropriate host cells for expression. Expression vectors foruse in carrying out the present invention will comprise a promotercapable of directing the transcription of a cloned DNA and atranscriptional terminator, operably linked with the sequence encodingthe human islet GAD so as to produce a continuously transcribable genesequence which produces sequences in reading frame and continuouslytranslated to produce a human islet GAD polypeptide.

Host cells for use in practicing the present invention includemammalian, avian, plant, insect, bacterial and fungal cells, butpreferably eukaryotic cells. Preferred eukaryotic cells include culturedmammalian cell lines (e.g., rodent or human cell lines, but mostpreferably human) and fungal cells, including species of yeast (e.g.,Saccharomyces spp., particularly S. cerevisiae, Schizosaccharomycesspp., or Kluyveromyces spp.) or filamentous fungi (e.g., Aspergillusspp., Neurospora spp.). Methods for producing recombinant polypeptidesin a variety of prokaryotic and eukaryotic host cells are generallyknown in the art.

To produce the recombinant human islet GAD polypeptides of the inventionin mammalian cells, a variety of host cells may be used. Culturedmammalian cells for use in the present invention include COS-1 (ATCC CRL1650), BALB/c 3T3 (ATCC CRL 163), BHK (ATCC CRL 10314), 293 (ATCC CRL1573), Rat Hep I (ATCC CRL 1600), Rat Hep II (ATCC CRL 1548), TCMK (ATCCCCL 139), Human lung (ATCC CCL 75.1), Human hepatoma (ATCC HTB-52), HepG2 (ATCC HB 8065), Mouse liver (ATCC CCL 29.1), NCTC 1469 (ATCC CCL 9.1)and DUKX cells (Urlaub and Chasin, Proc. Natl. Acad. Sci. USA 77:4216-4220, 1980). Beta-cell lines, such as those of rat (RIN-5AH-B;Karlsen, et. al., J. Biol. Chem. 266:7542-7548, (1991)), mouse (NIT;Hamaguchi et al., Diabetes 39:415-425 (1990)) and hamster (HIT;Santerre, et. al., Proc. Natl. Acad. Sci. USA 78:4339-4343 (1981)) mayalso be used.

Mammalian expression vectors for use in carrying out the presentinvention will include a promoter capable of directing the transcriptionof a cloned gene or cDNA. Preferred promoters include viral promotersand cellular promoters. Viral promoters include the immediate earlycytomegalovirus promoter (Boshart et al., Cell 41: 521-530, 1985), theSV40 promoter (Subramani et al., Mol. Cell. Biol. 1: 854-864, 1981), andthe major late promoter from Adenovirus 2 (Kaufman and Sharp, Mol. Cell.Biol. 2: 1304-1319, 1982). Cellular promoters include the mousemetallothionein-1 promoter (Palmiter et al., U.S. Pat. No. 4,579,821), amouse V.sub.κ promoter (Bergman et al., Proc. Natl. Acad. Sci. USA 81:7041-7045, 1983; Grant et al., Nuc. Acids Res. 15: 5496, 1987) and amouse V_(H) promoter (Loh et al., Cell 33: 85-93, 1983). Also containedin the expression vectors is a polyadenylation signal located downstreamof the coding sequence of interest. Polyadenylation signals include theearly or late polyadenylation signals from SV40 (Kaufman and Sharp,ibid.), the polyadenylation signal from the Adenovirus 5 E1B region andthe human growth hormone gene terminator (DeNoto et al., Nuc. Acids Res.9: 3719-3730, 1981). Vectors can also include enhancer sequences, suchas the SV40 enhancer and the mouse μ enhancer (Gillies, Cell 33:717-728, 1983). Expression vectors may also include sequences encodingthe adenovirus VA RNAs. Vectors can be obtained from commercial sources(e.g., Stratagene, La Jolla, Calif.).

Cloned DNA sequences may be introduced into cultured mammalian cells bya variety of means, as will be recognized by those skilled in the art.For example, calcium phosphate-mediated transfection (Wigler et al.,Cell 14: 725, 1978; Corsaro and Pearson, Somatic Cell Genetics 7: 603,1981; Graham and Van der Eb, Virology 52: 456, 1973), electroporation(Neumann et al., EMBO J. 1: 841-845, 1982), or DEAE-dextran mediatedtransfection (Ausubel et al., (ed.) Current Protocols in MolecularBiology, John Wiley and Sons, Inc., New York (1987), incorporated hereinby reference) may find convenient use. To identify cells that havestably integrated the cloned DNA, a selectable marker is generallyintroduced into the cells along with the gene or cDNA of interest.Preferred selectable markers for use in cultured mammalian cells includegenes that confer resistance to drugs, such as neomycin, hygromycin, andmethotrexate. Further, the selectable marker may be an amplifiableselectable marker, and preferred amplifiable selectable markers includethe DHFR gene and the neomycin resistance gene. Selectable markers arereviewed by Thilly (Mammalian Cell Technology, Butterworth Publishers,Stoneham, Mass., which is incorporated herein by reference). The choiceof selectable markers is well within the level of ordinary skill in theart.

Selectable markers may be introduced into the cell on a separate vectorat the same time as the islet cell GAD sequence of interest, or they maybe introduced on the same vector. If on the same vector, the selectablemarker and the islet cell GAD sequence of interest may be under thecontrol of different promoters or the same promoter, the latterarrangement producing a dicistronic message. Constructs of this type areknown in the art (for example, Levinson and Simonsen, U.S. Pat. No.4,713,339). It may also be advantageous to add additional DNA, known as"carrier DNA" to the mixture which is introduced into the cells.

Transfected mammalian cells are allowed to grow for a period of time,typically 1-2 days, to begin expressing the DNA sequence(s) of interest.Drug selection is then applied to select for growth of cells that areexpressing the selectable marker in a stable fashion. For cells thathave been transfected with an amplifiable selectable marker the drugconcentration may be increased in a stepwise manner to select forincreased copy number of the cloned sequences, thereby increasingexpression levels.

Promoters, terminators and methods for introducing expression vectorsencoding foreign proteins such as human islet GAD into plant, avian andinsect cells are well known in the art. The use of baculoviruses, forexample, as vectors for expressing heterologous DNA sequences in insectcells has been reviewed by Atkinson et al. (Pestic. Sci. 28:215-224,1990). The use of Agrobacterium rhizogenes as vectors forexpressing genes in plant cells has been reviewed by Sinkar et al. (J.Biosci. (Bangalore) 11: 47-58, 1987).

Techniques for transforming fungi are well known in the literature, andhave been described, for instance, by Beggs (Nature 275:104-108 (1978)),Hinnen et al. (Proc. Natl. Acad. Sci. USA 75: 1929-1933, 1978), Yeltonet al. (Proc. Natl. Acad. Sci. USA 81: 1740-1747, 1984), Russell (Nature301: 167-169, 1983) and U.S. Pat. No. 4,935,349, incorporated herein byreference. The genotype of the host cell will generally contain agenetic defect that is complemented by the selectable marker present onthe expression vector. Choice of a particular host and selectable markeris well within the level of ordinary skill in the art.

Suitable yeast vectors for use in the present invention include YRp7(Struhl et al., Proc. Natl. Acad. Sci. USA 76: 1035-1039, 1978), YEp13(Broach et al., Gene 8: 121-133, 1979), POT vectors (Kawasaki et al,U.S. Pat. No. 4,931,373, which is incorporated by reference herein),pJDB249 and pJDB219 (Beggs, ibid.) and derivatives thereof. Such vectorswill generally include a selectable marker, which may be one of anynumber of genes that exhibit a dominant phenotype for which a phenotypicassay exists to enable transformants to be selected. Preferredselectable markers are those that complement host cell auxotrophy,provide antibiotic resistance or enable a cell to utilize specificcarbon sources, and include LEU2 (Broach et al., ibid.), URA3 (Botsteinet al., Gene 8: 17, 1979), HIS3 (Struhl et al., ibid.) or POT1 (Kawasakiet al., ibid.). Another suitable selectable marker is the CAT gene,which confers chloramphenicol resistance on yeast cells.

Preferred promoters for use in yeast include promoters from yeastglycolytic genes (Hitzeman et al., J. Biol. Chem. 255: 12073-12080,1980; Alber and Kawasaki, J. Mol. Appl. Genet. 1: 419-434, 1982;Kawasaki, U.S. Pat. No. 4,599,311) or alcohol dehydrogenase genes (Younget al., in Genetic Engineering of Microorganisms for Chemicals,Hollaender et al., (eds.), p. 355, Plenum, New York, 1982; Ammerer,Meth. Enzymol. 101: 192-201, 1983). In this regard, particularlypreferred promoters are the TPIl promoter (Kawasaki, U.S. Pat. No.4,599,311, 1986) and the ADH2-4^(C) promoter (Russell et al., Nature304: 652-654, 1983; Irani and Kilgore, U.S. patent application Ser. No.183,130, which is incorporated herein by reference). The expressionunits may also include a transcriptional terminator. A preferredtranscriptional terminator is the TPI1 terminator (Alber and Kawasaki,ibid.).

Additional vectors, promoters and terminators which can be used inexpressing the human islet GAD of the invention in yeast are well knownin the art and are reviewed by, for example, Emr, Meth. Enzymol.185:231-279, (1990), incorporated herein by reference.

Host cells containing DNA constructs of the present invention are thencultured to produce the human islet cell GAD polypeptides. The cells arecultured according to standard methods in a culture medium containingnutrients required for growth of the chosen host cells. A variety ofsuitable media are known in the art and generally include a carbonsource, a nitrogen source, essential amino acids, vitamins and minerals,as well as other components, e.g., growth factors or serum, that may berequired by the particular host cells. The growth medium will generallyselect for cells containing the DNA construct by, for example, drugselection or deficiency in an essential nutrient which is complementedby the selectable marker on the DNA construct or co-transfected with theDNA construct. Selection of a medium appropriate for the particular cellline used is within the level of ordinary skill in the art.

For instance, yeast cells are preferably cultured in a medium whichcomprises a nitrogen source (e.g., yeast extract), inorganic salts,vitamins and trace elements. The pH of the medium is preferablymaintained at a pH greater than 2 and less than 8, preferably at pH 5-6.Methods for maintaining a stable pH include buffering and constant pHcontrol, preferably through the addition of sodium hydroxide. Preferredbuffering agents include succinic acid and Bis-Tris (Sigma Chemical Co.,St. Louis, Mo.). Cultured mammalian cells are generally cultured incommercially available serum-containing or serum-free media.

The human islet cell GAD produced according to the present invention maybe purified by affinity chromatography on an antibody column usingantibodies, preferably monoclonal antibodies, directed against GAD.Additional purification may be achieved by conventional chemicalpurification means, such as liquid chromatography, gradientcentrifugation, and gel electrophoresis, among others. Methods ofprotein purification are known in the art (see generally, Scopes, R.,Protein Purification, Springer-Verlag, New York (1982), which isincorporated herein by reference) and may be applied to the purificationof the recombinant human islet GAD described herein. Substantially purerecombinant human islet GAD of at least about 50% is preferred, at leastabout 70-80% more preferred, and 95-99% or more homogeneity mostpreferred, particularly for pharmaceutical uses. Once purified,partially or to homogeneity, as desired, the recombinant human islet GADmay then be used diagnostically, therapeutically, etc. as furtherdescribed herein below.

Human islet GAD polypeptides can also be produced by fragmenting largerpurified recombinant GAD polypeptides with a protease or a chemicalagent, or by producing recombinant polypeptide fragments. Syntheticislet cell GAD peptides can also be produced from the amino acidsequences provided herein, using conventional solid-phase synthesisprocedures as described in, e.g., Merrifield, Fed. Proc. 21:412 (1962)and Barany and Merrifield, in The Peptides, Vol. 2, pp. 1-284 (1979)Academic Press, New York, which are incorporated herein by reference.Short polypeptide sequences, or libraries of overlapping peptides,usually from about 6 up to about 35 amino acids, which correspond toselected human islet GAD regions can be readily synthesized and thenscreened in screening assays designed to identify peptides having adesired activity, such as domains which are responsible for orcontribute to GAD catalytic activity, binding activity, immunodominantepitopes (particularly those recognized by autoantibodies), and thelike. Recombinant polypeptides can be produced by expressing GAD DNAfragments, such as fragments generated by digesting a human islet cellGAD cDNA at convenient restriction sites. The isolated recombinantpolypeptides or cell-conditioned media are then assayed for activity asdescribed above.

Human islet cell GAD polypeptides produced according to the presentinvention have a variety of uses. For example, recombinant or syntheticGAD polypeptide compositions can be used diagnostically, in thedetection and quantitation of anti-GAD autoantibodies or detecting freeGAD (as a measure of beta cell destruction) in a biological sample, thatis, any sample derived from or containing cells, cell components or cellproducts, including, but not limited to, cell culture supernatants, celllysates, cleared cell lysates, cell extracts, tissue extracts, blood,plasma, serum, and fractions thereof. By means of having human islet GADpolypeptides which specifically bind to human islet GAD autoantibodies,the concentration of the autoantibodies in an individual can bemeasured, which level can then be used to monitor the progression orregression of the potentially harmful autoantibodies in individuals atrisk. The assay results can also find use in monitoring theeffectiveness of therapeutic measures for treatment of IDDM, Stiff-manSyndrome, or related diseases.

As will be recognized by those skilled in the art, numerous types ofimmunoassays are available for use in the present invention. Forinstance, direct and indirect binding assays, competitive assays,sandwich assays, and the like, as are generally described in, e.g., U.S.Pat. Nos. 4,642,285; 4,376,110; 4,016,043; 3,879,262; 3,852,157;3,850,752; 3,839,153; 3,791,932; and Harlow and Lane, Antibodies, ALaboratory Manual, Cold Spring Harbor Publications, New York (1988),each incorporated by reference herein. In one assay format anti-humanislet GAD autoantibodies in a biological sample are quantified directlyby measuring the binding of antibodies to recombinant or synthetic GADpolypeptides. The biological sample is contacted with at least one humanislet cell GAD polypeptide of the invention under conditions conduciveto immune complex formation. The immune complexes formed between the GADpolypeptide and the antibodies are then detected and the presence of theautoantibodies to human islet cell GAD in the sample determined. Theimmune complexes can be detected by means of, e.g., labeled antibodies,such as anti-IgG, IgM and/or IgA human antibodies, or antibodies whichbind to the human GAD. Separation steps (e.g., washes) may be necessaryin some cases to distinguish specific binding over background. Inanother format, a patient's antibodies or serum GAD can be measured bycompeting with labeled or unlabeled antibodies to GAD or GADpolypeptides, respectively, for binding. Unlabeled GAD may be used incombination with labeled antibodies which bind to human antibodies or toGAD. Alternatively, the GAD polypeptide may be directly labeled. A widevariety of labels may be employed, such as radionuclides, particles(e.g., gold, ferritin, magnetic particles, red blood cells), fluors,enzymes, enzyme substrates, enzyme cofactors enzyme inhibitors, ligands(particularly haptens), chemiluminescers, etc.

Thus, autoantibodies to β-islet cell GAD autoantigens may be identifiedand, if desired, extracted from patient's serum by binding to the GAD.The GAD polypeptide may be attached, e.g., by absorption, to aninsoluble or solid support, such as an ELISA microtiter well,microbeads, filter membrane, insoluble or precipitable soluble polymer,etc. to function as an affinity resin. The captured autoantibodies maythen be identified by several methods. For example, antisera ormonoclonal antibodies to the antibodies may be used. These antisera ormonoclonal antibodies are typically non-human in origin, such as rabbit,goat, mouse, etc. These anti-antibodies may be detected directly ifattached to a label such as ¹²⁵ I, enzyme, biotin, etc., or may bedetected indirectly by a labeled secondary antibody made to specificallydetect the anti-antibody.

Kits can also be supplied for use with the recombinant or synthetichuman islet GAD polypeptides in detecting autoantibodies to pancreaticβ-islet cells. Thus, the subject GAD polypeptide compositions of thepresent invention may be provided, usually in lyophilized form, in acontainer, either alone or in conjunction with additional reagents, suchas GAD-specific antibodies, labels, and/or anti-human antibodies, andthe like. The GAD polypeptide and antibodies, which may be conjugated toa label, or unconjugated, and are included in the kits with buffers,such as Tris, phosphate, carbonate, etc., stabilizers, biocides, inertproteins, e.g., serum albumin, or the like. Frequently it will bedesirable to include an inert extender or excipient to dilute the activeingredients, where the excipient may be present in from about 1 to 99%of the total composition. Where an antibody capable of binding to theislet GAD autoantibody or to the recombinant or synthetic GAD isemployed in an assay, this will typically be present in a separate vial.

Antibodies for diagnostic or therapeutic uses which bind human islet GADand/or islet cell GAD polypeptides of the invention can be produced by avariety of means. The production of non-human monoclonal antibodies,e.g., murine, is well known and may be accomplished by, for example,immunizing the animal with a recombinant or synthetic GAD molecule or aselected portion thereof (e.g., a peptide). For example, by selectedscreening one can identify a region of the GAD molecule such as thatpredominantly responsible for recognition by anti-GAD autoantibodies, ora portion which comprises an epitope of an islet cell GAD variableregion, which may thus serve as an islet cell GAD-specific marker.Antibody producing cells obtained from the immunized animals areimmortalized and screened, or screened first for, e.g., the productionof antibody which inhibits the interaction of the anti-GAD autoantibodywith the GAD molecule and then immortalized. As the generation of humanmonoclonal antibodies to a human antigen, such as the human islet cellGAD molecule, may be difficult with conventional immortalizationtechniques, it may be desirable to first make non-human antibodies andthen transfer via recombinant DNA techniques the antigen binding regionsof the non-human antibodies, e.g. the F(ab')₂ or hypervariable regions,to human constant regions (Fc) or framework regions to producesubstantially human molecules. Such methods are generally known in theart and are described in, for example, U.S. Pat. No. 4,816,397, EPpublications 173,494 and 239,400, which are incorporated herein byreference. Alternatively, one may isolate DNA sequences which encode ahuman monoclonal antibody or portions thereof that specifically bind tothe human islet cell GAD protein by screening a DNA library from human Bcells according to the general protocol outlined by Huse et al., Science246:1275-1281 (1989), incorporated herein by reference, and then cloningand amplifying the sequences which encode the antibody (or bindingfragment) of the desired specificity.

Antibodies which bind to the recombinant human islet cell GAD, includingthose antibodies which inhibit the binding of autoantibodies to thehuman islet cell GAD autoantigen can also be used in the generation ofanti-idiotype antibodies. Anti-idiotype antibodies may be produced by,for example, immunizing an animal with the primary antibody or with theantigen binding fragment thereof. Those anti-idiotype antibodies whosebinding to the primary antibody is inhibited by the human islet cell GADmolecule are selected. Since both the anti-idiotype antibody and the GADmolecule bind to the primary antibody, the anti-idiotype antibody mayrepresent the "internal image" of an epitope and thus may substitute forthe GAD autoantigen and inhibit disease by blocking the GADautoantibodies. Treatment of autoimmune diseases with intravenousimmunoglobulin preparations containing anti-idiotype antibodies isdisclosed by, for example, Nydegger, et. al., (Clin. Immunol.Immunopathol. 53 (2 part 2):572-82 (1989)).

In another aspect of the invention, the human islet GAD polypeptides ofthe invention can be used to clone T cells which have specific receptorsfor GAD molecules. Once the GAD-specific T cells are isolated and clonedusing techniques generally available to the skilled artisan, the T cellsor membrane preparations thereof can be used to immunize animals toproduce antibodies to the islet GAD receptors on T cells. The antibodiescan be polyclonal or monoclonal. If polyclonal, the antibodies and canbe murine, lagomorph, equine, ovine, or from a variety of other mammals.Monoclonal antibodies will typically be murine in origin, producedaccording to known techniques, or human, as described above, orcombinations thereof, as in chimeric or humanized antibodies. Theanti-GAD receptor antibodies thus obtained can then be administered topatients to reduce or eliminate T cell subpopulations which recognizeand participate in the immunological destruction of GAD-bearing cells inan individual predisposed to or already suffering from the disease.Further, the GAD-specific T cell receptors can thus be identified,cloned and sequenced, and receptor polypeptides synthesized which bindto the GAD molecules and block recognition of the GAD-bearing cells,thereby impeding the autoimmune response against host islet cells.Howell et al. (Science 246:668-670 (1989)) have demonstrated that T cellreceptor peptides can block the formation of the tri-molecular complexbetween T cells, autoantigen and major histocompatibility complex in anautoimmune disease model.

In other embodiments the invention concerns human islet GAD polypeptideswhich inhibit the binding of autoantibodies to human GAD islet cellautoantigen and inhibit the proliferation of T cells. The portion(s) ofthe GAD autoantigen protein which binds the auto-antibodies may beidentified using relatively short fragments of GAD islet polypeptides,as generally described above, and determining which fragment(s) binds tothe autoantibodies, particularly those autoantibodies associated withIDDM or related disease, such as those antibodies isolated from apatient using plasmapheresis as discussed below.

Thus, in another aspect of the invention the human islet cell GADpolypeptides can be used in immunoadsorptive plasmapheresis therapy toremove autoantibodies from the circulation of an individual. Anindividual undergoing such treatment will typically have detectablelevels of anti-GAD autoantibodies and thus will be at risk of developingdisease associated with such autoantibodies, such as IDDM or Stiff-mansyndrome, or will already be afflicted by such disease. Theplasmapheresis treatment is provided by removing the patient's blood,separating the blood cells therefrom, treating the separated plasma in,e.g., an immunoadsorbent column to remove the autoantibodies, and mixingand returning the treated plasma and blood cells directly to thepatient. Typically the patient's blood is removed, treated and returnedto the patient in a continuous manner.

The immunoadsorbent column for treating the plasma will comprise arecombinant islet cell GAD as described herein, covalently coupled to asolid-phase matrix. Means for coupling polypeptides to varioussolid-phase matrices are generally known in the art. Typically thecovalent coupling, which can include a "spacer" molecule, as describedin e.g., U.S. Pat. No. 4,685,900, which is incorporated herein byreference, is accomplished by appropriately derivatizing the solid-phasematrix and linking the protein under conditions which maximize thebinding activity and capacity of the GAD polypeptide of the invention.Desirably, the immunoadsorbent thus formed has a high capacity foradsorption of the autoantibodies, is highly stable and is not releasedinto the plasma. The volume of plasma which is treated and the frequencyof treatment will depend on, e.g., the severity of the disease beingtreated, the quantity of autoantibodies in a patient's plasma, theoverall health and condition of the patient, and the judgment of theattending physician. In any event, the treatment should be sufficient toprevent or alleviate the symptoms or arrest development of the disease.

The human islet cell GAD polypeptides of the invention can also be usedto induce immunological tolerance or nonresponsiveness (anergy) to GADautoantigen in patients predisposed or already mounting an immuneresponse to GAD autoantigen of the islet β-cells. The use of polypeptideantigens in the suppression of autoimmune disease is disclosed byWraith, et. al., (Cell 59:247-255, (1989)). Tolerance can be induced inboth adults and neonates, although conditions for inducing suchtolerance will vary according to a variety of factors. In a neonate,tolerance can be induced by parenteral injection of GAD antigen, eitherwith recombinant polypeptide or synthetic antigen, or more convenientlyby oral administration in an appropriate formulation. The precise amountof administration, its mode and frequency of dosages will vary.

To induce immunological tolerance to the GAD autoantigen in an adultsusceptible to or already suffering from a GAD related disease such asIDDM, the precise amounts and frequency of administration will alsovary, but for adults will generally range from about 1 to 1,000 mg/kg,preferably about 10-100 mg/kg, administered daily or from one to severaltimes per week, and will be administered by a variety of routes, such asparenterally, orally, by aerosol, intradermal injection, etc., butpreferably by intravenous infusion. For neonates the doses willgenerally be higher than those administered to adults; e.g., typicallyfrom about 100 to 1,000 mg/kg. To induce tolerance with lower doses ofantigen (a "low zone tolerance") co-administration of animmunosuppressive drug, such as cyclophosphamide or, preferred for usein children, azathioprine, may be necessary during the low dose antigentreatment to inhibit antibody synthesis.

The GAD polypeptides will typically be more tolerogenic whenadministered in a soluble form rather than an aggregated or particulateform. Persistence of a GAD polypeptide antigen of the invention isgenerally needed to maintain tolerance in an adult, and thus may requiremore frequent administration of the antigen, or its administration in aform which extends the half-life of the GAD islet cell polypeptide.

The following examples are offered by way of illustration, not bylimitation.

EXAMPLE I Cloning and Sequencing of Human Islet Cell GAD

Islet cells were isolated from human pancreata obtained from organtransplant donors for whom a matched recipient was not available. Afterin situ perfusion with cold UW solution (Du Pont, Boston, Mass.), eachpancreas was carefully excised, the pancreatic duct cannulated, and 4mg/ml collagenase solution (Type V, Sigma, St. Louis, Mo.) infused at aconstant rate, first at 4° C. and then 39° C. The gland was teasedapart, and liberated fragments were washed by centrifugation, trituratedthrough needles of decreasing caliber, and purified by discontinuousFicoll density centrifugation (G. L. Warnock, Diabetes 35: Suppl. 1, pp.136-139, Jan. 1989). Material harvested from the upper interfaces waspooled and counted after a determination of islet purity by dithiazonestaining. Islets used in library construction were greater than 65%pure, while islets used in Northern blots were greater than 40% pure.The average islet diameter was 175 μm. Additionally, the isolated isletsshowed both first and second phase insulin secretory function afterperfusion with either high glucose or with isobutylmethylxanthine(IBMX).

Poly(A)⁺ RNA was isolated using the FastTrack™ mRNA isolation kit(Invitrogen, San Diego, Calif.) according to the manufacturer'sinstructions. Briefly, 30,000 purified islets were quickly lysed inlysis buffer, homogenized using needles of decreasing caliber, anddigested in the presence of proteinase K and RNasin, then poly(A)⁺ RNAwas selected by oligo-d(T) cellulose chromatography. The concentrationand purity of the eluted fractions were determined at OD_(260/280).

Approximately 2.5 μg poly(A)⁺ RNA from the human islets was used forcDNA library construction using a Librarian R II cDNA libraryconstruction system (Invitrogen) and Electromax™ DH10B E. coli cells(GIBCO BRL, Gaithersburg, Md.) according to the manufacturers'instructions. In short, approximately 2.5 μg of poly(A)⁺ RNA, isolatedfrom human islets, was converted into double-stranded cDNA, followed bythe addition of BstX I nonpalindromic linkers (Invitrogen). The cDNA wassize fractionated, and the unreacted linkers were removed by agarose gelelectrophoresis and electroelution. Complementary DNA strands largerthan 600 bp were selected and ligated into the Librarian R II pcDNA IIvector. Following electroporation of a fraction of the ligated materialinto DH 10B E. coli cells, a total of 2×10⁶ colonies with a backgroundof approximately 10% was screened by hybridization. These colonies werereplicated in duplicate to nylon filters, lysed, neutralized, washed andbaked essentially as described by Sambrook et al., Molecular Cloning. ALaboratorv Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.(1989), incorporated herein by reference.

To identify colonies containing human islet GAD cDNA, 20-meroligonucleotide probes representing conserved regions from threedifferent homologous nucleotide sequences of the internal as well as theN- and C- terminal parts of the coding regions of cat (Kobayashi, et al.J. Neurosci. 7:2768-2772 (1987)), rat (Julien et al., J. Neurochem.54:703-705 (1990)), and mouse (Katarova, et al. ibid.) brain GAD weresynthesized (Table 1), ³² P-ATP labeled by kinasing and used to screenthe nylon filter replicas of the human islet cDNA library. Followinghybridization and consecutive washings at increasing stringency, sixpositive colonies representing insert sizes from 0.7 to 1.4 kb wereselected for colony purification and subsequent sequence analysis. Byrescreening the library with a 600 bp PvuII-PstI fragment of a circa1300 bp clone containing 1281 base pairs of the 3' coding sequence(pHIG1.3), another clone, pHIG1.9, FIG. 1, with a 1.9 kb insert, wasisolated.

                                      TABLE 1                                     __________________________________________________________________________     ##STR1##                                                                      ##STR2##                                                                      ##STR3##                                                                     __________________________________________________________________________

For DNA sequencing, plasmids were isolated from positive clones by therapid boiling method (Homes and Quigley, Anal. Biochem. 114:193-197(1981). The resulting double-stranded cDNA was sequenced using theSequenase® kit (version 2.0, United States Biochemical, Cleveland,Ohio.) with primers hybridizing to the flanking SP6 and T7 promoters. Asthe sequencing progressed, new primers representing 20-mer 3'oligonucleotides of the insert were synthesized and used to obtain theentire sequence of the human islet GAD cDNA inserts. The nucleotidesequences were analyzed with the sequence analysis software package ofthe University of Wisconsin genetics computer group (Devereux, Nuc.Acids. Res. 12:387-396, (1984), incorporated herein by reference).

To obtain full-length clones, the 5' CDNA ends of two clones wereextended using a variation of the PCR-RACE protocol (Frohman et al.,Proc. Natl. Acad. Sci. USA 85: 8998-9002, 1988). Oligonucleotide primers(ZC3614, ZC3623, Table 2) complementary to a region near the 5' end ofGAD clones, and containing adapter sequences with an Eco RI restrictionsite, were annealed with an aliquot of islet cell poly(A)⁺ RNA.Following extension, the products were terminaldeoxynucleotidyltransferase-tailed with dGTP, and a poly-dCTP primerwith an Eco RI adapter (ZC2488, Table 2) was used on second strandsynthesis to generate a cDNA population enriched for GAD but stillheterogeneous due to non-specific pairing of the internal primer. As asecond step, a second oligonucleotide complementary to a region upstreamfrom the internal primer (ZC3746 upstream of ZC3614 or ZC3745 upstreamof ZC3623; Table 2), was used to prime the minus strand while a primercomplementary to the Eco RI adapter (ZC2633; Table 2) was used to primethe plus strand. PCR amplification yielded further enrichment of GADsequences. Using the GeneAmp PCR kit (Perkin-Elmer Cetus, Norwalk,Conn.), the reaction was cycled 40 times at 94° C. for 1 minute todenature, 50° C. for 2 minutes and 72° C. for 2 minutes. The resultingproducts were electrophoresed on an agarose gel, and the GAD sequenceswere eluted, digested with Eco RI and cloned into the vector pUC19.

                                      TABLE 2                                     __________________________________________________________________________     ##STR4##                                                                     __________________________________________________________________________

Double-stranded plasmids containing the 5' end of the GAD sequenceinserted into pUCl9 were sequenced in the same manner as the positiveclones initially isolated from the library using the primers in Table 3.

                  TABLE 3                                                         ______________________________________                                         ##STR5##                                                                     ______________________________________                                    

The entire sequence of the human islet cell GAD cDNA is shown in FIG. 2,SEQ. ID. NO. 1. It was determined by assembling a composite of twooverlapping cDNA clones, pHIG 11 and pHIG1.9, and of five RACE reactionproducts, RACE 20, 28A, 47A, 41 and 42 (FIG. 1). The two cDNA clonesoverlap by 110 bp and the five RACE sequences were designed to overlapwith pHIG 1.9 by 140 nucleotides at the 3' end (FIG. 1). The pHIG 1.9clone comprises 1900 bp and encodes the pyridoxal 5'-phosphate bindingsite (Pro-His-Lys-Met-Met-Gly) at amino acids 394-399, a stop codonfollowing the C-terminal Leu codon and a polyadenylation site (AAATAAA),17 nucleotides upstream of a poly A sequence at the 3' end (FIG. 2).pHIG 11 extended the 5' end of the cDNA sequence to 250 bp upstream fromthe predicted N-terminal Met. pHIG1.1 contains an intron as a cloningintron from an aberrantly spliced RNA.

The human islet GAD cDNA nucleotide sequence had a overall homology ofonly about 70% to the brain GAD cDNA sequences of the rat, cat andmouse, whereas the brain sequences showed about 91% homology among thethree. The predicted amino acid sequence homology between the humanislet GAD and those of the brain sequences of cat, rat and mouse isabout 76%, again in contrast to the more than about 98% homology foundamong the brain GAD amino acid sequences. Comparison of the 5'-end ofthe human islet sequence to the 68 amino acid 5'-end of the human testisGAD (Perrson et al., Mol. Cell. Biol. 10:4701-4711 (1990)) showsdifferences at 15 amino acid positions, whereas only two amino acidsubstitutions have been found between the human testis sequence and ratbrain GAD.

A hydropathy plot of the human islet GAD primary structure indicatedregions of increased hydrophobicity when compared to rat and cat brainGAD (FIG. 3). Apart from N-terminal end differences, the sequences atamino acid positions 165-195 were particularly different. The hydropathyplot analysis suggests that the human islet GAD has features which aredistinct from the rat and mouse brain GAD sequences.

EXAMPLE II Tissue Expression and Chromosomal Location of Human Islet GAD

Northern blot analysis was used to detect human islet GAD mRNAexpression in normal human islets as well as in different in vitrocultured beta-cell lines. Beta-cell lines from rat (RIN-5AH-B), mouse(NIT cells) and hamster (HIT) cells of low passage number were culturedat 37° C. in 150 cm² tissue culture flasks in RPMI 1640 mediumsupplemented with 10% (v/v) heat-inactivated fetal calf serum, 20 mmol/lHepes, 2 mmol/l L-glutamine, 24 mmol/l NaHCO₃, 100 U/ml penicillin and100 μg/ml streptomycin (RPMI medium). The results were obtained withcells plated in a standardized manner at a density of 23×10³ cells percm². After 3 days to allow establishment of the culture, the medium wasrenewed. At this timepoint, referred to as day 0, the cell density wasapproximately 1×10⁵ cells per cm². Over the next 4 days (days 1-4) thecells were collected for analysis of GAD gene expression by Northernblotting. The cells were detached after washing in Ca²⁺ - and Mg²⁺ -freeEarle's medium by incubation at 37° C. for 5 min. in Earle's mediumsupplemented with 0.25% (w/v) trypsin (GIBCO BRL, Gaithersburg, Md.) and1 mmol/l EDTA. After detachment, the cells were washed and immediatelylysed for mRNA isolation as described in Example I above.

Northern blot analysis was performed as follows. About 5 μg poly A⁺ RNA,estimated at A₂₆₀, was heated to 95° C. for 2 min. and separated by 1%agarose formaldehyde gel electrophoresis. Following separation, theagarose gel was washed twice for 20 min. in 10× saline-sodium-citratebuffer (SSC), the RNA blotted overnight onto a nitrocellulose filter(Scheicher & Schuell, Inc., Keene, New Hampshire) and the filter bakedfor 2 h at 80° C. as described (Davis, et al., Basic Methods Molec. BiolElsevier, Amsterdam, 1986). The cDNA or oligonucleotide probes asdescribed above were used after nick translation (GIBCO BRL) accordingto instructions provided by the manufacturer. Following 4 hprehybridization at 42° C., fresh hybridization buffer containing thesingle-stranded probe at about 10⁶ CPM/ml was added to the filter andhybridized overnight at 42° C. as described (Davis, et al., ibid). Thefilter was washed 3× for 20 min. at room temperature in 2× SSCcontaining 0.1% SDS and 2× for 20 min. in 2× SSC containing 0.1% SDS at42° C. before it was exposed to X-ray film (X-omat XAR, Eastman Kodak,Rochester, N.Y.). Before reprobing, the filter was washed 3×5 min. in0.2× SSC, 0.1% SDS at 95° C.

Northern blot analysis revealed a pronounced 5.7 kb transcript in polyA⁺ RNA isolated from islets of dog and rat, as well as from brain ofdog, monkey and rat. Expression of the human islet GAD was not detectedin RIN-5AH cells, AL-34 cells, rat liver, muscle, testis and kidney.Reprobing the Northern blots with a probe representing the rat brain GADcDNA revealed a 3.7 kb transcript not only in dog and rat brain, butalso in the RIN cells. No cross hybridization to the 5.6 kb transcriptwas detected.

Genomic DNA from cells of patients with IDDM and from healthyindividuals were probed in Southern blots with human islet GAD cDNA. Thegenomic DNA (20 μg) was digested with different restriction enzymesfollowed by electrophoretic separation on agarose gels. The patternobtained with the human islet GAD cDNA probe was clearly different fromthat obtained with rat brain GAD cDNA probe, suggesting that thefragment patterns were generated by different genes.

EXAMPLE III Detection of GAD Autoantibodies

The GAD cDNA was inserted into the vector pcDNAII (Invitrogen, SanDiego, Calif.) to construct pEx9 and transcribed in vitro. A reactionmixture was prepared by combining 20 μl of 5× SP6 transcription buffer(GIBCO BRL); 10 μl of 100 mM DTT; 100 units RNAsin; 7.5 μl of 2.5 mMeach ATP, CTP and UTP; 2.5 μl of 1 mM GTP; 5 μl of 5 mM m7GpppG (capanalog); 2 μg linearized pEx9 DNA; 2 μl SP6 polymerase (GIBCO BRL) anddistilled water to a final volume of 100 μl. The reaction mixture wasincubated for 90 minutes at 37° C. The mixture wasphenol-chloroform-isoamylalcohol extracted and then ethanolprecipitated. The RNA pellet was resuspended in distilled water to afinal concentration of 1 mg/ml.

The resulting synthetic mRNA was subjected to in vitro translation with³⁵ S-methionine in a rabbit reticulocyte lysate system. The in vitrotranslation (IVT) reaction mixture contained 35 μl nuclease-treatedrabbit reticulocyte lysate (Promega, Madison, Wis.), 50 units RNAsin, 1μl amino acid mix (-Met), 1 μl ³⁵ S-methionine at a concentration of150Ci/mmol and 50mCi/ml, and distilled water to a volume of 48 μl. TheSP6 RNA, prepared as described above, was denatured at 67° C. for 10minutes and then placed on ice. One microgram of the denatured SP6 RNAin a final volume of 2 μl was added to the IVT reaction mixture. Thereaction mixture was incubated at 30° C. for 90 minutes. Two microlitersof the reaction was precipitated with TCA to calculate percentincorporation as described by Sambrook et al. (ibid). The in vitrosynthesized product represented a single Mr 64,000 protein.

The labeled, synthesized protein was used to screen sera for thepresence of GAD autoantibodies. Protein A-Sepharose immunoprecipitationshowed that sera from ten newly diagnosed IDDM children precipitated11.6±2.9% (mean ±SEM) of the total radioactivity, compared with 2.3±0.5%in 22 healthy controls (p<0.001). Gel electrophoresis andautoradiography revealed that healthy controls remained negative while8/10 IDDM sera precipitated the in vitro-synthesized protein. Thespecific immunoprecipitation with IDDM sera indicates that the majorautoepitope is likely present on the nascent polypeptide and does notrequire post-translational modifications by an intact cell.

In a similar experiment, in vitro-synthesized, ³⁵ S-methionine-labeledGAD was used in an overnight radioligand binding assay using proteinA-Sepharose to separate bound from free ligand. IMP buffer was preparedusing 150 mM NaCl, 20 mM Tris pH 7.4, 1% Triton X-100, 0.1% Aprotinin,and 10 mM Benzamidine. A high salt IMP buffer was prepared with 400 mMNaCl substituted for the 150 mM NaCl. Forty-seven and one-halfmicroliters of IMP buffer was added to 0.5 μl in vitro translated GADand 2 μl of sera. The mixture was incubated by rotating overnight at 4°C. The Protein A-Sepharose was prewashed with IMP buffer and aliquotedinto tubes at 50 μl/tube. The tubes were rotated for 1 hour at 4° C. TheProtein A-Sepharose was then washed three times with 400 μl IMP buffer,one time with 400 μl high salt IMP buffer and one time with 400 μl IMPbuffer. One hundred microliters of 1× SDS sample buffer, without anydyes, was added to each tube, and the mixture was boiled for 10 minutes.The supernatants were removed, added to 4 ml of scintillation fluid andcounted. GAD antibody-positive (the Juvenile Diabetes Foundation serumfor ICA standardization) and negative control sera were included in eachassay to express autoantibody levels as a 64K index. A 64K index isdefined as:

    mean cpm (sample).sup.˜ mean cpm (negative control) mean cpm (positive control).sup.˜ mean cpm (negative control)

The intra-assay coefficient of variation for duplicate determinationswas 10.5%. In 38 0-15 year old controls the 64 K index was -0.031±0.007(mean ±SEM). In 62 new onset, 0-15 year old IDDM patients, the 64K indexwas 0.48±0.082. At a dilution of 1:25, the IDDM sera precipitated11.7±1.6% of the total ligand radioactivity compared to 1.9±0.1% in thecontrols (p<0.001). Using a 64K index of 0.03 as the upper level ofnormal, no control (0/38) was positive compared to 48/62 (77%) of theIDDM patients. The 64K index in IDDM correlated to levels of ICA (r_(s)=0.58; p<0.001). Thus, autoantibodies against synthetic human islet GADcan be accurately detected in a radioligand assay and are closelyassociated with newly diagnosed IDDM in children.

EXAMPLE IV Expression of GAD cDNA

Expression of the human islet GAD cDNA required that the two overlappingclones, pHIG11 and pHIG1.9 (Seq. ID. No. 1) be assembled into a singlefull length clone. The 5' sequence from the open reading frame of clonepHIGll was isolated using a polymerase chain reaction. Oligonucleotideswere synthesized so the 5' end of one primer was positioned at the ATGinitiation codon and contained the following sequence:

    5' CCA GTC TGA ATT CAC CAT GCT AGC CCA GGC TCC GGA T 3'    (Seq. ID. No. 14)

The 3' oligonucleotide primer began at a NsiI sits, 482 nucleotidesdownstream of the initiation codon, and contained the followingsequence:

    5' TTT TAG AGA AGC TTG GCA ATG CAT CAA AAT TTC CTC C 3'    (Seq. ID. No. 15).

A 0.5 kb DNA fragment was isolated by digestion at the EcoRI (5') andHindIII (3') restriction sites, and after sequence analysis was found tobe the correct size. The EcoRI site was altered to a BamHI site usingthe synthetic oligonucleotide 5' ATT GGA TCC 3'. The resulting 0.5 kbDNA fragment was then isolated using the BamHI (5') and the NaiI (3')restriction sites.

The remaining DNA sequence of the GAD cDNA was isolated as 2 cDNAfragments from the clone designated pHIG1.9 (Seq. ID. No. 1). Theinternal DNA fragment was isolated using the restriction enzymes NsiI(5') and BglII (3'). The resulting fragment was found to be 0.6 kb. The3' end of the GAD cDNA sequence was isolated by digestion of the clonepHIG1.9 with the restriction enzymes BglII (5') and XbaI (3'), and theresulting fragment was found to be 0.72 kb.

The expression plasmid was made from a four-part ligation reaction thatincluded the 5' BamHI-NsiI fragment, the internal NsiI-BglII fragment,the 3' BglII-XbaI fragment and the expression vector Zem 219b.

The vector Zem 219b was constructed in the following manner. PlasmidpIC19R (Marsh et al., Gene 32:481-486 (1984)) was digested with SmaI andHind III. The ori region of SV40 from map position 270 (PvuII) toposition 5171 (HindIII) was then ligated to the linearized pIC19R toproduce plasmid Zem67. This plasmid was then cleaved with Bgl II, andthe terminator region from the human growth hormone gene (De Noto etal., Nuc. Acids Res. 9: 3719-3730 (1980)) was inserted as a Bgl II-BamHI fragment to produce plasmid Zem86. A synthesized human plasminogenactivator (t-PA) pre-pro sequence in pUC8 was isolated by digestion withBamHI and XhoII. This fragment was inserted into Bgl II digested Zem86to produce plasmid Zem88. Plasmid pDR1296 (ATCC 53347) was digested withBgl II and BamH I, and the t-PA cDNA fragment was isolated and insertedinto Bgl II-cut Zem88. The resultant plasmid was designated Zem94. Thevector Zem99, comprising the MT-1 promoter, complete t-PA codingsequence, and the human growth hormone (hGH) terminator, was thenassembled. A KpnI-BamHI fragment comprising the MT-1 promoter wasisolated from MThGH111 (Palmiter et al., Science 222:809-814 (1983)) andinserted into pUC18 to construct Zem93. Plasmid EV142, comprising MT-1and hGH sequences in the pBR322 derivative pBX322 (Palmiter et al.,ibid.), was digested with Eco RI, and the fragment comprising the MT-1promoter and hGH terminator sequences was isolated. This fragment wascloned into Eco RI-digested pUC13 to construct plasmid Zem4. Zem93 wasthen linearized by digestion with Bgl II and Sal I, and the hGHterminator was purified. The t-PA pre-pro sequence was removed from thepUC8 vector as a Sau 3A fragment. The three DNA fragments were thenjoined to produce plasmid Zem97. Zem97 was cut with Bgl II and the BglII-BamH I t-PA fragment from pDR1296 was inserted. The resultant vectorwas designated Zem99. Plasmid pSV2-DHFR (Subramani et al., ibid.) wasdigested with Cfo I, and the fragment containing the DHFR cDNA and the3' attached SV40 sequences was isolated, repaired, and ligated to BamHIlinkers. After digestion with BamH I, an approximately 800 bp fragmentcontaining the entire cDNA and the SV40 terminator region was purifiedand ligated to BamHI-digested PUC8. Zem67 was digested with Bgl II andligated with the BamHI DHFR-SV40 fragment to generate plasmid Zem176.Plasmid Zem93 was digested with Sst I and re-ligated to generate plasmidZem106, in which approximately 600 bp of sequence 5' to the MT-1promoter was eliminated. Plasmid Zem106 was digested with Eco RI andligated to the EcoRI fragment containing the DHFR gene from plasmidZem176. The resulting plasmid was designated Zts13. Plasmid Zts13 wasdigested with BamHI and ligated to the BamHI fragment from plasmid Zem99containing the entire t-PA coding region and hGH terminator sequence.The resulting plasmid was designated Zts15. Zts15 was partially digestedwith BamHI, repaired and re-ligated to generate plasmid Zem219, in whichthe 3' BamHI site was destroyed. Plasmid Zem219 was partially digestedwith XbaI, repaired and re-ligated to generate plasmid Zem219a, in whichthe XbaI site 3' to the hGH terminator was destroyed. Zem219b wasderived from Zem219a by digesting that vector with BamHI and XbaI,removing the t-PA sequences, and ligating the vector fragment with aBamHI-XbaI adaptor. Zem219b has been deposited with American TypeCulture Collection, Rockville, Md. as an E. coli XL1-blue transformant.

Expression of the GAD cDNA was achieved by transfection of the tk⁻ ts13BHK cell line (ATCC CRL 1632) using the calcium phosphate method (Vander Eb, ibid.). Transfectants were selected using a medium containing400 nM methotrexate. The transfectants were tested for production ofhuman GAD protein using immunocytochemistry. For testingimmunoreactivity, two antibodies were used (Michelsen et al., Proc.Natl. Acad. Sci. USA 88:8754-8758 (1991)). The first antibody,designated 1266, was raised in rabbits immunized with the syntheticC-terminal sequence:

    Thr-Gln-Ser-Asp-Ile-Asp-Phe-Leu-Ile-Glu-Glu-Ile-Glu-Arg-Leu-Gly-Gln-Asp-Leu(Seq. ID. No. 16)

The second antibody used for immunofluorescence labeling was raisedagainst GABA (Immunotech, Marseille, France). The two-color doubleimmunofluorescence labeling was carried out on fixed (1%paraformaldehyde, neutral) monolayers of transfected BHK cells to testthe co-localization of the immunoreactivities of the C-terminalantiserum 1266 and antiserum against GABA. Texas Red-goat anti-rabbitIgG (1:100 dilution; Axell (Westbury, N.Y.)) was used to detect primaryantibodies. These assays showed that BHK cells transfected with humanGAD cDNA expressed immunoreactive material while host cells without theGAD cDNA did not demonstrate reactivity.

The recombinant GAD protein was purified from confluent cultures of9.5×10⁸ transfected cells. The cells were pelleted by centrifugation at350×g at room temperature for 4 minutes. The resulting cell pellet washomogenized in 20 ml of 50 mM sodium phosphate, 1 mM pyridoxal5'-phosphate (PLP), 1 mM amino-ethyl-isothiouronium-bromide (AET), 1 mMEDTA, 0.05% W/V aprotinin, 1% W/V Triton X-114 (TX-114) pH 8.0 (bufferA) and shaken gently for 1 hour at 4° C. for 30 minutes. Once insuspension the mixture was centrifugated at 100,000×g at 4° C. for 30minutes. Twenty milliters of supernant was poured on top of 20 ml of 6%(w/v) sucrose, and the mixture was heated to 30° C. for 3 minutes.Following incubation the mixture was centrifuged at 3290×g for 5minutes. The aqueous phase was extracted as described previously byadding 0.5% w/v TX-114 and applied to the same sucrose fraction as usedpreviously. Nine ml of buffer A without TX-114 was added to the 1 mldetergent phase. The diluted TX-114 detergent phase of 10 ml was appliedto a 1.0×1.6 cm GAD-1-Sepharose affinity column. GAD-1 is a monoclonalantibody against the human GAD protein. The column was washed in 40 mlof buffer A. The sample was applied to the column a total of threetimes. After the final application the column was washed with 70 ml of50 mM sodium phosphate, 1 mM PLP, 1 mM AET, 0.05% aprotinin and 1% w/vn-octyl glucoside pH 8.0 (buffer B). The GAD was eluted with 50 mM NH₄HCO₃, 1% w/v n-octyl glucoside, 1 mM PLP and 1 mM AET pH 9.5 andcollected in 500 μl fractions. Five hundred microliters of 50 mM sodiumphosphate pH 7.0 was added to each fraction. The column was washed inbuffer B and stored in PBS and 0.02% NaN₃. Ten microliter aliquots ofthe first ten fractions were analyzed using 1D-SDS polyacrylamide gelelectrophoresis. Using a polyclonal antibody raised against theC-terminus of the human GAD protein, western analysis was done. Inaddition, the gel was stained with Coomassie brilliant blue, andenzymatic activity was measured as described in Wu (Methods inEnzymology 113:3-10 (1985)). Pools 4-14 were combined, and the totalprotein yield was calculated to be 27 μg or 27 ng/10⁶ BHK cells. Theprotein purity was evaluated using a 2D-PAGE analysis, which showed amajor band at 64 kD.

The foregoing provides isolated and purified human islet cell GADnucleotide sequences and recombinant human islet GAD polypeptides. Theseresults offer, inter alia, a reproducible system to detectautoantibodies to the Mr 64,000 autoantigen in sera from patients withinsulin-dependent diabetes or individuals with subclinical disease.Oligonucleotide probes for detecting the islet cell sequences in situare also provided by means of the present invention, as well astherapeutic approaches to preventing or alleviating diseases related toan autoimmune response to the human islet GAD antigen.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be apparent that certain changes andmodifications may be practiced within the scope of the appended claims.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 16                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2370 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 38..1792                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GGCACTCGCTGGCGACCTGCTCCAGTCTCCAAAGCCGATGGCATCTCCGGGCTCT55                     MetAlaSerProGlySer                                                            15                                                                            GGCTTTTGGTCTTTCGGGTCGGAAGATGGCTCTGGGGATTCCGAGAAT103                           GlyPheTrpSerPheGlySerGluAspGlySerGlyAspSerGluAsn                              101520                                                                        CCCGGCACAGCGCGAGCCTGGTGCCAAGTGGCTCAGAAGTTCACGGGC151                           ProGlyThrAlaArgAlaTrpCysGlnValAlaGlnLysPheThrGly                              253035                                                                        GGCATCGGAAACAAACTGTGCGCCCTGCTCTACGGAGACGCCGAGAAG199                           GlyIleGlyAsnLysLeuCysAlaLeuLeuTyrGlyAspAlaGluLys                              404550                                                                        CCGGCGGAGAGCGGCGGGAGCCAACCCCCGCGGGCCGCCGCCCGGAAG247                           ProAlaGluSerGlyGlySerGlnProProArgAlaAlaAlaArgLys                              55606570                                                                      GCCGCCTGCGCCTGCGACCAGAAGCCCTGCAGCTGCTCCAAAGTGGAT295                           AlaAlaCysAlaCysAspGlnLysProCysSerCysSerLysValAsp                              758085                                                                        GTCAACTACGCGTTTCTCCATGCAACAGACCTGCTGCCGGCGTGTGAT343                           ValAsnTyrAlaPheLeuHisAlaThrAspLeuLeuProAlaCysAsp                              9095100                                                                       GGAGAAAGGCCCACTTTGGCGTTTCTGCAAGATGTTATGAACATTTTA391                           GlyGluArgProThrLeuAlaPheLeuGlnAspValMetAsnIleLeu                              105110115                                                                     CTTCAGTATGTGGTGAAAAGTTTCGATAGATCAACCAAAGTGATTGAT439                           LeuGlnTyrValValLysSerPheAspArgSerThrLysValIleAsp                              120125130                                                                     TTCCATTATCCTAATGAGCTTCTCCAAGAATATAATTGGGAATTGGCA487                           PheHisTyrProAsnGluLeuLeuGlnGluTyrAsnTrpGluLeuAla                              135140145150                                                                  GACCAACCACAAAATTTGGAGGAAATTTTGATGCATTGCCAAACAACT535                           AspGlnProGlnAsnLeuGluGluIleLeuMetHisCysGlnThrThr                              155160165                                                                     CTAAAATATGCAATTAAAACAGGGCATCCTAGATACTTCAATCAACTT583                           LeuLysTyrAlaIleLysThrGlyHisProArgTyrPheAsnGlnLeu                              170175180                                                                     TCTACTGGTTTGGATATGGTTGGATTAGCAGCAGACTGGCTGACATCA631                           SerThrGlyLeuAspMetValGlyLeuAlaAlaAspTrpLeuThrSer                              185190195                                                                     ACAGCAAATACTAACATGTTCACCTATGAAATTGCTCCAGTATTTGTG679                           ThrAlaAsnThrAsnMetPheThrTyrGluIleAlaProValPheVal                              200205210                                                                     CTTTTGGAATATGTCACACTAAAGAAAATGAGAGAAATCATTGGCTGG727                           LeuLeuGluTyrValThrLeuLysLysMetArgGluIleIleGlyTrp                              215220225230                                                                  CCAGGGGGCTCTGGCGATGGGATATTTTCTCCCGGTGGCGCCATATCT775                           ProGlyGlySerGlyAspGlyIlePheSerProGlyGlyAlaIleSer                              235240245                                                                     AACATGTATGCCATGATGATCGCACGCTTTAAGATGTTCCCAGAAGTC823                           AsnMetTyrAlaMetMetIleAlaArgPheLysMetPheProGluVal                              250255260                                                                     AAGGAGAAAGGAATGGCTGCTCTTCCCAGGCTCATTGCCTTCACGTCT871                           LysGluLysGlyMetAlaAlaLeuProArgLeuIleAlaPheThrSer                              265270275                                                                     GAACATAGTCATTTTTCTCTCAAGAAGGGAGCTGCAGCCTTAGGGATT919                           GluHisSerHisPheSerLeuLysLysGlyAlaAlaAlaLeuGlyIle                              280285290                                                                     GGAACAGACAGCGTGATTCTGATTAAATGTGATGAGAGAGGGAAAATG967                           GlyThrAspSerValIleLeuIleLysCysAspGluArgGlyLysMet                              295300305310                                                                  ATTCCATCTGATCTTGAAAGAAGGATTCTTGAAGCCAAACAGAAAGGG1015                          IleProSerAspLeuGluArgArgIleLeuGluAlaLysGlnLysGly                              315320325                                                                     TTTGTTCCTTTCCTCGTGAGTGCCACAGCTGGAACCACCGTGTACGGA1063                          PheValProPheLeuValSerAlaThrAlaGlyThrThrValTyrGly                              330335340                                                                     GCATTTGACCCCCTCTTAGCTGTCGCTGACATTTGCAAAAAGTATAAG1111                          AlaPheAspProLeuLeuAlaValAlaAspIleCysLysLysTyrLys                              345350355                                                                     ATCTGGATGCATGTGGATGCAGCTTGGGGTGGGGGATTACTGATGTCC1159                          IleTrpMetHisValAspAlaAlaTrpGlyGlyGlyLeuLeuMetSer                              360365370                                                                     CGAAAACACAAGTGGAAACTGAGTGGCGTGGAGAGGGCCAACTCTGTG1207                          ArgLysHisLysTrpLysLeuSerGlyValGluArgAlaAsnSerVal                              375380385390                                                                  ACGTGGAATCCACACAAGATGATGGGAGTCCCTTTGCAGTGCTCTGCT1255                          ThrTrpAsnProHisLysMetMetGlyValProLeuGlnCysSerAla                              395400405                                                                     CTCCTGGTTAGAGAAGAGGGATTGATGCAGAATTGCAACCAAATGCAT1303                          LeuLeuValArgGluGluGlyLeuMetGlnAsnCysAsnGlnMetHis                              410415420                                                                     GCCTCCTACCTCTTTCAGCAAGATAAACATTATGACCTGTCCTATGAC1351                          AlaSerTyrLeuPheGlnGlnAspLysHisTyrAspLeuSerTyrAsp                              425430435                                                                     ACTGGAGACAAGGCCTTACAGTGCGGACGCCACGTTGATGTTTTTAAA1399                          ThrGlyAspLysAlaLeuGlnCysGlyArgHisValAspValPheLys                              440445450                                                                     CTATGGCTGATGTGGAGGGCAAAGGGGACTACCGGGTTTGAAGCGCAT1447                          LeuTrpLeuMetTrpArgAlaLysGlyThrThrGlyPheGluAlaHis                              455460465470                                                                  GTTGATAAATGTTTGGAGTTGGCAGAGTATTTATACAACATCATAAAA1495                          ValAspLysCysLeuGluLeuAlaGluTyrLeuTyrAsnIleIleLys                              475480485                                                                     AACCGAGAAGGATATGAGATGGTGTTTGATGGGAAGCCTCAGCACACA1543                          AsnArgGluGlyTyrGluMetValPheAspGlyLysProGlnHisThr                              490495500                                                                     AATGTCTGCTTCTGGTACATTCCTCCAAGCTTGCGTACTCTGGAAGAC1591                          AsnValCysPheTrpTyrIleProProSerLeuArgThrLeuGluAsp                              505510515                                                                     AATGAAGAGAGAATGAGTCGCCTCTCGAAGGTGGCTCCAGTGATTAAA1639                          AsnGluGluArgMetSerArgLeuSerLysValAlaProValIleLys                              520525530                                                                     GCCAGAATGATGGAGTATGGAACCACAATGGTCAGCTACCAACCCTTG1687                          AlaArgMetMetGluTyrGlyThrThrMetValSerTyrGlnProLeu                              535540545550                                                                  GGAGACAAGGTCAATTTCTTCCGCATGGTCATCTCAAACCCAGCGGCA1735                          GlyAspLysValAsnPhePheArgMetValIleSerAsnProAlaAla                              555560565                                                                     ACTCACCAAGACATTGACTTCCTGATTGAAGAAATAGAACGCCTTGGA1783                          ThrHisGlnAspIleAspPheLeuIleGluGluIleGluArgLeuGly                              570575580                                                                     CAAGATTTATAATAACCTTGCTCACCAAGCTGTTCCACTTCTCTAGAGA1832                         GlnAspLeu                                                                     585                                                                           ACATGCCCTCAGCTAAGCCCCCTACTGAGAAACTTCCTTTGAGAATTGTGCGACTTCACA1892              AAATGCAAGGTGAACACCACTTTGTCTCTGAGAACAGACGTTACCAATTATGGAGTGTCA1952              CCAGCTGCCAAAATCGTAGGTGTTGGCTCTGCTGGTCACTGGAGTAGTTGCTACTCTTCA2012              GAATATGGACAAAGAAGGCACAGGTGTAAATATAGTAGCAGGATGAGGAACCTCAAACTG2072              GGTATCATTTGCACGTGCTCTTCTGTTCTCAAATGCTAAATGCAAACACTGTGTATTTAT2132              TAGTTAGGTGTGCCAAACTACCGTTCCCAAATTGGTGTTTCTGAATGACATCAACATTCC2192              CCCAACATTACTCCATTACTAAAGACAGAAAAAAATAAAAACATAAAATATACAAACATG2252              TGGCAACCTGTTCTTCCTACCAAATATAAACTTGTGTATGATCCAAGTATTTTATCTGTG2312              TTGTCTCTCTAAACCCAAATAAATGTGTAAATGTGGACACAAAAAAAAAAAAAAAAAA2370                (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 585 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       MetAlaSerProGlySerGlyPheTrpSerPheGlySerGluAspGly                              151015                                                                        SerGlyAspSerGluAsnProGlyThrAlaArgAlaTrpCysGlnVal                              202530                                                                        AlaGlnLysPheThrGlyGlyIleGlyAsnLysLeuCysAlaLeuLeu                              354045                                                                        TyrGlyAspAlaGluLysProAlaGluSerGlyGlySerGlnProPro                              505560                                                                        ArgAlaAlaAlaArgLysAlaAlaCysAlaCysAspGlnLysProCys                              65707580                                                                      SerCysSerLysValAspValAsnTyrAlaPheLeuHisAlaThrAsp                              859095                                                                        LeuLeuProAlaCysAspGlyGluArgProThrLeuAlaPheLeuGln                              100105110                                                                     AspValMetAsnIleLeuLeuGlnTyrValValLysSerPheAspArg                              115120125                                                                     SerThrLysValIleAspPheHisTyrProAsnGluLeuLeuGlnGlu                              130135140                                                                     TyrAsnTrpGluLeuAlaAspGlnProGlnAsnLeuGluGluIleLeu                              145150155160                                                                  MetHisCysGlnThrThrLeuLysTyrAlaIleLysThrGlyHisPro                              165170175                                                                     ArgTyrPheAsnGlnLeuSerThrGlyLeuAspMetValGlyLeuAla                              180185190                                                                     AlaAspTrpLeuThrSerThrAlaAsnThrAsnMetPheThrTyrGlu                              195200205                                                                     IleAlaProValPheValLeuLeuGluTyrValThrLeuLysLysMet                              210215220                                                                     ArgGluIleIleGlyTrpProGlyGlySerGlyAspGlyIlePheSer                              225230235240                                                                  ProGlyGlyAlaIleSerAsnMetTyrAlaMetMetIleAlaArgPhe                              245250255                                                                     LysMetPheProGluValLysGluLysGlyMetAlaAlaLeuProArg                              260265270                                                                     LeuIleAlaPheThrSerGluHisSerHisPheSerLeuLysLysGly                              275280285                                                                     AlaAlaAlaLeuGlyIleGlyThrAspSerValIleLeuIleLysCys                              290295300                                                                     AspGluArgGlyLysMetIleProSerAspLeuGluArgArgIleLeu                              305310315320                                                                  GluAlaLysGlnLysGlyPheValProPheLeuValSerAlaThrAla                              325330335                                                                     GlyThrThrValTyrGlyAlaPheAspProLeuLeuAlaValAlaAsp                              340345350                                                                     IleCysLysLysTyrLysIleTrpMetHisValAspAlaAlaTrpGly                              355360365                                                                     GlyGlyLeuLeuMetSerArgLysHisLysTrpLysLeuSerGlyVal                              370375380                                                                     GluArgAlaAsnSerValThrTrpAsnProHisLysMetMetGlyVal                              385390395400                                                                  ProLeuGlnCysSerAlaLeuLeuValArgGluGluGlyLeuMetGln                              405410415                                                                     AsnCysAsnGlnMetHisAlaSerTyrLeuPheGlnGlnAspLysHis                              420425430                                                                     TyrAspLeuSerTyrAspThrGlyAspLysAlaLeuGlnCysGlyArg                              435440445                                                                     HisValAspValPheLysLeuTrpLeuMetTrpArgAlaLysGlyThr                              450455460                                                                     ThrGlyPheGluAlaHisValAspLysCysLeuGluLeuAlaGluTyr                              465470475480                                                                  LeuTyrAsnIleIleLysAsnArgGluGlyTyrGluMetValPheAsp                              485490495                                                                     GlyLysProGlnHisThrAsnValCysPheTrpTyrIleProProSer                              500505510                                                                     LeuArgThrLeuGluAspAsnGluGluArgMetSerArgLeuSerLys                              515520525                                                                     ValAlaProValIleLysAlaArgMetMetGluTyrGlyThrThrMet                              530535540                                                                     ValSerTyrGlnProLeuGlyAspLysValAsnPhePheArgMetVal                              545550555560                                                                  IleSerAsnProAlaAlaThrHisGlnAspIleAspPheLeuIleGlu                              565570575                                                                     GluIleGluArgLeuGlyGlnAspLeu                                                   580585                                                                        (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (vii) IMMEDIATE SOURCE:                                                       (B) CLONE: ZC3338                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       GCGGGAGCGGATCCTAATACTACCAACCTGCG32                                            (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (vii) IMMEDIATE SOURCE:                                                       (B) CLONE: ZC3339                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       ACCATGGTTGTTCCTGACTCCATCAT26                                                  (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 45 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (vii) IMMEDIATE SOURCE:                                                       (B) CLONE: ZC3337                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       CTGACATCAACTGCCAATACCAATATGTTCACATATGAAATTGCA45                               (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (vii) IMMEDIATE SOURCE:                                                       (B) CLONE: ZC3614                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       AAATGAGAATTCACACGCCGGCAGCAGGTC30                                              (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (vii) IMMEDIATE SOURCE:                                                       (B) CLONE: ZC3623                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       AAGGAATTCAAGTTGATTGAAGTATCT27                                                 (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (vii) IMMEDIATE SOURCE:                                                       (B) CLONE: ZC3745                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       GGCGAATTCGCATATTTTAGAGTTGTTTGG30                                              (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (vii) IMMEDIATE SOURCE:                                                       (B) CLONE: ZC3746                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       GGCGAATTCGGAGCAGCTGCAGGGCTTCTG30                                              (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (vii) IMMEDIATE SOURCE:                                                       (B) CLONE: ZC2633                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      AGGGAGACCGGAATTCGACTCGAGTCGACATCGATCAG38                                      (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      GACTCGAGTCGACATCGATCAGCCCCCCCCCC32                                            (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      GGCGATTAAGTTGGGTAA18                                                          (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      TAACAATTTCACACAGG17                                                           (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      CCAGTCTGAATTCACCATGCTAGCCCAGGCTCCGGAT37                                       (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      TTTTAGAGAAGCTTGGCAATGCATCAAAATTTCCTCC37                                       (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE:                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      ThrGlnSerAspIleAspPheLeuIleGluGluIleGluArgLeuGly                              151015                                                                        GlnAspLeu                                                                     __________________________________________________________________________

What is claimed is:
 1. A method for determining the presence ofautoantibodies to human islet cell glutamic acid decarboxylase (GAD) ina biological sample, the method comprising:contacting the biologicalsample with a recombinant human islet cell GAD polypeptide free of otherhuman proteins and produced by a cell transformed or transfected with aconstruct for expressing said GAD polypeptide, which construct comprisesa DNA sequence encoding said GAD polypeptide wherein said sequencecomprises the nucleotide sequence of FIG. 2, SEQ ID No. 1, fromnucleotide 38 to nucleotide 1792, or a nucleotide sequence degeneratethereto which codes for the GAD polypeptide encoded by said nucleotidesequence of FIG. 2, SEQ ID No. 1, from nucleotide 38 to nucleotide 1792,wherein the polypeptide catalyzes the synthesis of γ-aminobutyric acid,and detecting the presence of immune complex formation between said GADpolypeptide and autoantibodies to human islet cell GAD and therefromdetermining the presence of autoantibodies to human islet cell GAD inthe sample.
 2. The method of claim 1, wherein the GAD polypeptide isattached to a solid phase support.
 3. The method of claim 1, wherein theGAD polypeptide is labeled.
 4. The method of claim 1, wherein saidimmune complex is detected by a second antibody.
 5. The method of claim1, wherein said detecting step is by enzyme reaction, fluorescence,luminescence, or radioactivity.
 6. The method of claim 1, wherein thebiological sample is blood, plasma, serum, or urine.